The Role of Sphingosine Kinase 1/Sphingosine-1- Phosphate ... · Funding: This study was supported by a faculty research grant from Yonsei University College of Medicine in 2010 (No

The Role of Sphingosine Kinase 1/Sphingosine-1-Phosphate Pathway in the Myogenic Tone of PosteriorCerebral ArteriesMihwa Lim, Soo-Kyoung Choi, Young-Eun Cho, Soo-In Yeon, Eok-Cheon Kim, Duck-Sun Ahn, Young-

Ho Lee*

Department of Physiology, College of Medicine, BK 21 Project for Medical Sciences, Yonsei University, Seoul, Korea

Abstract

Aims: The goal of the current study was to determine whether the sphingosine kinase 1 (SK1)/sphingosine-1-phosphate(S1P) pathway is involved in myogenic vasoconstriction under normal physiological conditions. In the present study, weassessed whether endogenous S1P generated by pressure participates in myogenic vasoconstriction and which signalingpathways are involved in SK1/S1P-induced myogenic response under normal physiological conditions.

Methods and Results: We measured pressure-induced myogenic response, Ca2+ concentration, and 20 kDa myosin lightchain phosphorylation (MLC20) in rabbit posterior cerebral arteries (PCAs). SK1 was expressed and activated by elevatedtransmural pressure in rabbit PCAs. Translocation of SK1 by pressure elevation was blocked in the absence of external Ca2+

and in the presence of mechanosensitive ion channel and voltage-sensitive Ca2+ channel blockers. Pressure-inducedmyogenic tone was inhibited in rabbit PCAs treated with sphingosine kinase inhibitor (SKI), but was augmented bytreatment with NaF, which is an inhibitor of sphingosine-1-phosphate phosphohydrolase. Exogenous S1P furtheraugmented pressure-induced myogenic responses. Pressure induced an increase in Ca2+ concentration leading to thedevelopment of myogenic tone, which was inhibited by SKI. Exogenous S1P further increased the pressure-inducedincreased Ca2+ concentration and myogenic tone, but SKI had no effect. Pressure- and exogenous S1P-induced myogenictone was inhibited by pre-treatment with the Rho kinase inhibitor and NADPH oxidase inhibitors. Pressure- and exogenousS1P-induced myogenic tone were inhibited by pre-treatment with S1P receptor blockers, W146 (S1P1), JTE013 (S1P2), andCAY10444 (S1P3). MLC20 phosphorylation was increased when the transmural pressure was raised from 40 to 80 mmHg andexogenous S1P further increased MLC20 phosphorylation. The pressure-induced increase of MLC20 phosphorylation wasinhibited by pre-treatment of arteries with SKI.

Conclusions: Our results suggest that the SK1/S1P pathway may play an important role in pressure-induced myogenicresponses in rabbit PCAs under normal physiological conditions.

Citation: Lim M, Choi S-K, Cho Y-E, Yeon S-I, Kim E-C, et al. (2012) The Role of Sphingosine Kinase 1/Sphingosine-1-Phosphate Pathway in the Myogenic Tone ofPosterior Cerebral Arteries. PLoS ONE 7(4): e35177. doi:10.1371/journal.pone.0035177

Editor: Ryuichi Morishita, Osaka University Graduate School of Medicine, Japan

Received January 11, 2012; Accepted March 9, 2012; Published April 20, 2012

Copyright: � 2012 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This study was supported by a faculty research grant from Yonsei University College of Medicine in 2010 (No. 6-2010-0056). The funders had no role instudy design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

The myogenic response is an intrinsic vascular response

characterized by vasoconstriction in response to an increase in

intravascular pressure and vasodilation in response to a decrease in

intravascular pressure [1]. Arterial myogenic tone plays an

important role in establishing ambient vascular tone and auto-

regulating blood flow in the resistance vasculature, especially in

cerebral circulation [2–4], because cerebral arteries are not

particularly responsive to the sympathetic nerves surrounding

them [5].

The biologically active sphingomyelin metabolite, sphingosine-

1-phosphate (S1P), generated by the enzyme sphingosine kinase 1

(SK1), is present in plasma at high nanomolar concentrations,

released from activated platelets [6,7], and found in increased

quantities in inflammation and atherosclerosis [8]. S1P plays an

important role as a vascular modulator [9–11], and most effects of

S1P are mediated by a family of five highly specific G-protein–

coupled receptors called S1P receptors [12]. It was reported that

the myogenic responses of isolated resistance arteries were

increased in the smooth muscle cells of SK1-transfected arteries

[12,13]. It was also reported that myogenic vasoconstriction in

response to increased transmural pressure was significantly

reduced in resistance arteries transfected with sphingosine-1-

phosphate phosphohydrolase 1 (SPP1), a S1P-degrading enzyme

[14]. Taken together, these results suggest that SK1 and its

product, S1P, may be involved in the pressure-induced signaling

cascade leading to myogenic vasoconstriction. However, whether

SK1/S1P contributes to pressure-induced myogenic responses

under normal physiological conditions is unknown.

PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35177

Cerebral vasospasm is a sustained abnormal contraction of the

cerebral arteries [15,16]. Several spasmogenic substances in-

cluding oxyhemoglobin, endothelin-1, thrombin, serotonin, nor-

adrenalin, and thromboxane have been suggested [17,18].

Sphingolipids have been suggested as candidate spasmogenic

substances because they are released from activated platelets and

are found at high levels in inflammation and predisposing

situations for vasospasm [17]. Therefore, the changes in vascular

contractility, including myogenic tone, induced by sphingolipids in

cerebral arteries should be determined. We already reported the

augmentation of cerebral arterial tone by sphingolipid metabolites

as well as the underlying mechanisms [19]. Therefore, the role of

the SK1/S1P pathway in pressure-induced myogenic tone and the

underlying mechanisms remain to be elucidated.

The goal of this study was to determine whether SK1/S1P

participates in pressure-induced myogenic responses under normal

physiological conditions and which signaling pathways are involved

in SK1/S1P-induced myogenic responses. We examined the effect

of endogenous and exogenous S1P on myogenic responses. We

measured the contribution of endogenous S1P on myogenic

response upon vasoconstriction induced by activated SK1 when

transmural pressure was increased, and the effect of exogenous S1P

was evaluated as vasoconstriction was induced by treatment with

S1P in a bath solution at elevated transmural pressure.

Methods

This investigation conformed to the Guide for the Care and Use of

Laboratory Animals published by the US National Institutes of

Health (NIH publication No. 85–23, revised in 1996). The

experimental protocols used in this study were reviewed and

approved by the Ethics Committee and Institutional Animal Care

and Use Committee of Yonsei University College of Medicine.

1.1. Tissue PreparationNew Zealand white rabbits were used in this study and

anesthetized with sodium pentobarbital (50 mg/kg, IV injection)

containing, heparin (2000 IU/kg), an anticoagulant. The rabbit

brains were removed and placed in a normal Krebs-Henseleit

(KH) solution composed of the following substances (in mmol/L):

NaCl, 119; CaCl2, 2.5; NaHCO3, 25; MgSO4, 1.2; KH2PO4, 1.2;

KCl, 4.6; and glucose, 11.1. The KH solution was continuously

aerated with a 95% O2-5% CO2 gas mixture. The rabbit posterior

cerebral arteries (PCAs) were dissected and their segments of about

3–4 mm in length were prepared.

1.2. Measurement of Myogenic Tone Using anArteriograph SystemRabbit PCA segments (100–250 mm inner diameter and 3–

4 mm in length) were dissected, cannulated in a pressure

myograph (Living Systems Instrumentation, Burlington, VT,

USA) filled with KH solution, and subsequently placed on the

stage of an inverted microscope (Eclipse TS100/TS100-F, Nikon

Inc., Melville, NY, USA). The proximal cannula was connected to

a solid-state pressure transducer and reservoir of KH solution, and

the transmural pressure was controlled by a pressure servo-

controller. The distal cannula was connected to a luer-lock valve

that was opened to flush the lumen during the initial cannulation.

After cannulation, the valve was closed, and all measurements

were conducted under no-flow conditions. The arterial lumen

diameter was recorded using the SoftEdge Acquisition Subsystem

(IonOptix, Milton, MA, USA).

The KH perfusing and superfusing in the arterial segments was

equilibrated with a 95% O2-5% CO2 gas mixture at 37uC. To

eliminate the potential influence of endothelial factors on the

pressure-induced myogenic tone, an air bolus was passed through

the lumen to disrupt the endothelium. The function of the

endothelium was checked at the beginning of each experiment

with 10–6 M acetylcholine in arteries pre-contracted with 70 mM

K+ solution (equimolar substitution of Na+ with K+).

After being mounted, the de-endothelialized cerebral arterial

segments were stretched longitudinally to approximate the in situ

length and were maintained at a transmural pressure of

40 mmHg for a 40–60 min equilibration period. After the

equilibration period, the pressure was increased in a stepwise

manner from 20 to 100 mmHg in 20-mmHg increments, and

each pressure was maintained for 10 min to allow blood vessel

diameter to stabilize before measurement. In some experiments,

an abbreviated myogenic protocol was used. The arteries were

equilibrated and constricted as described above, but the initial

pressure was 40 mmHg and was increased to 80 mmHg. After

a series of step changes, the transmural pressure was returned to

40 mmHg and the vessel was allowed to re-equilibrate for

a minimum of 40 min. At the end of each experiment, a passive

pressure-diameter relationship was established in Ca2+-free KH

solution containing 0.1 mmol/L nifedipine to determine the

maximum passive diameter. When the effects of drugs on

myogenic responses were assessed, they were administered at

a transmural pressure of 40 mmHg 15–30 min before increasing

the luminal pressure.

Responses to changes in transmural pressure were normalized

as a percentage of the initial diameter at 40 mmHg to control for

changes in the resting tone caused by the drugs. The following

formula was used to calculate the percent myogenic tone at each

pressure step: percent myogenic tone = {(DpX/Dp40) 2 (DaX/

Da40)}6100, where DpX and Dp40 are the passive diameters at

a given pressure and 40 mmHg in Ca2+-free PSS (0 mM Ca2+

with 0.1 mmol/L nifedipine), respectively, and DaX and Da40 are

the active diameters at a given pressure and 40 mmHg in normal

PSS in the presence of extracellular Ca2+, respectively.

1.3. Measurement of Smooth Muscle Ca2+ in PressurizedArteriesRabbit PCA segments were loaded with the Fura-2AM

(10 mmol/L; Molecular Probes, Eugene, OR, USA), the Ca2+-

sensitive fluorescent indicator, and 0.02% cremophor EL (Sigma,

St Louis, MO, USA) in KH as previously reported [20]. The

arteries were incubated in this solution for 3 h at room

temperature in the dark. Fura-2AM-loaded PCA segments were

mounted in a pressure myograph and pressurized to 40 mmHg

using a pressure-servo controller and then superfused with KH

(37uC) that was aerated with 95% O2 + 5% CO2 to wash out the

excess dye and to allow for hydrolysis of the AM groups by

intracellular esterases. The transmural pressure of the arteries was

then elevated to 80 mmHg. Fura-2-loaded vessels were alternately

excited at 340 and 380 nm at a frequency of 1 Hz with an

IonOptix Hyperswitch dual excitation light source, and the

respective emissions at 510 nm were detected with a photomulti-

plier tube. Background-subtracted 340/380 emission ratios were

calculated using IonOptix Ion Wizard software and recorded

continuously throughout the experiment. The fluorescent emission

at 510 nm (R340/380) and the changes in arterial diameter,

monitored by video microscopy (IonOptix), were recorded

simultaneously.

1.4. Immunofluorescence of SK1 in PCAThe localization of SK1 in isolated rabbit PCAs was measured

using immunostaining as described previously [21]. PCAs were

Role of SK1/S1P in the Myogenic Tone of Rabbit PCA


probed with SK1 antibody (1:200, Millipore, Temecula, CA,

USA).

1.5. Western BlottingThe expression of SK1 and S1P receptor proteins in rabbit

PCAs were measured by western blot as described previously [19].

Membranes were probed with SK1 antibody (1:200, Millipore),

S1P receptors (1:200, Millipore), and actin antibody (1:5,000,

Abcam, Cambridge, UK) was used as a loading control.

1.6. MLC20 Phosphorylation MeasurementsPhosphorylation of the 20 kDa myosin light chain (MLC20) in

the rabbit PCA was measured as described previously [19]. Vessels

were rapidly removed from the pressure myograph and frozen

when myogenic responses were stable after changes in transmural

pressure from 40 mmHg to 80 mmHg. For each preparation,

vessels from three to four animals were pooled. Membranes were

probed with a specific phosphor-MLC20 monoclonal antibody

(1:100; Cell Signaling, Boston, MA, USA) and total MLC20

antibody (1:200; Cell Signaling).

1.7. DrugsThe following drugs were used: S1P (Sigma), SKI (EIPA; Sigma,

St Louis, MO, USA), NaF (Sigma), (R)-3-Amino-(3-hexylpheny-

lamino)-4-oxobutylphosphonic acid (W146) (Sigma), 1-[1,3-Di-

methyl-4-(2-methylethyl)-1H-pyrazolo[3,4-b]pyridine-6-yl]-4-(3,5-

dichloro-4-pyridinyl)-semicarbazide (JTE013) (Tocris Bioscience,

Ellisville, MO, USA), 2-undecyl-thiazolidine-4-carboxylic acid

(CAY10444) (Cayman Chemical, Ann Arbor, MI, USA), fasudil

hydrochloride (Tocris), DPI (Sigma), apocynin (Sigma), 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hy-

drochloride (SKF-96365) (Tocris Bioscience), 9-phenanthrol

(Sigma), and nifedipine (Sigma). The general laboratory reagents

used were analytical grade or better. S1P was dissolved in 100%

ethanol. SKF96365 and 9-phenanthrol were dissolved in dimethyl

sulfoxide (DMSO) to a maximum final DMSO concentration of

0.1% that had no effect on vascular tone (data not shown).

1.8. StatisticsAll values given in the text were expressed as mean6SEMs and

analyzed by one-way and two-way ANOVA, followed by the

Student-Newman-Keuls post-hoc test. Differences were consid-

ered significant at the P,0.05 level.

Results

2.1. Activation of SK1 by Transmural Pressure ElevationWe first determined whether SK1 is expressed in rabbit PCAs

using western blot analysis. The western blots used to detect SK1

expression in isolated rabbit PCAs are shown in Figure 1A. SK1

antibodies were labeled as a major band around 50 kDa in isolated

PCAs, basilar arteries (BAs), middle cerebral arteries (MCAs), and

internal carotid arteries (ICAs).

We also determined whether SK1 is activated by pressure

elevation. Elevation of transmural pressure from 40 to 80 mmHg

was associated with translocation of SK1 from the cytosol to the

plasma membrane (Figure 1B). Translocation of SK1 by pressure

elevation was blocked in the absence of external Ca2+ (Ca2+-free

KH solution; Figure 1C1). Translocation of SK1 by pressure

elevation was also blocked in the presence of mechanosensitive ion

channel blockers, 1 mmol/L amiloride (epithelial Na+ channel

blocker), 5 mmol/L 9-phenanthrol (selective TRPM4 blocker), and

10 mmol/L SKF 96365 (nonselective blocker of TRPC channels)

(Figure 1C2). Nifedipine, a voltage-sensitive Ca2+ channel blocker,

also blocked translocation of SK1 by elevation of transmural

pressure from 40 to 80 mmHg (Figure 1C3).

2.2. Role of Endogenous and Exogenous S1P in MyogenicToneTo determine whether endogenous S1P generated by trans-

mural pressure elevation is involved in the development of

myogenic tone, we evaluated myogenic tone in the presence of

5 mmol/L SKI, an inhibitor of SK1 (Figures 2A & B1), and

1 mmol/L NaF, an inhibitor of the S1P-degrading enzyme,

sphingosine-1 phosphate phosphohydrolase (SPP) (Figure 2B2).

The myogenic response induced by increased transmural pressure

(stepwise from 20 to 100 mmHg) was decreased in the presence of

SKI (n= 9), but it was increased in the presence of NaF (n= 6).

To determine the effect of exogenous S1P on myogenic tone, we

evaluated myogenic tone in isolated rabbit PCA in the presence

and absence of exogenous S1P (1 mmol/L; Figure 2B3). The

myogenic response elicited by a pressure increase from 20 to

100 mmHg was elevated in the presence of exogenous S1P (n= 5).

We also determined the effect of pre-treatment of S1P on SKI-

induced inhibitory effect of myogenic tone. As shown in Figure 2C,

the SKI inhibited myogenic responses induced by increased

transmural pressure (from 40 to 80 mmHg). Pre-treatment of S1P

decreased blood vessel diameter and abolished the SKI-induced

inhibitory effect of myogenic tone. These results suggest that an

increase in transmural pressure activates SK1, which then

increases endogenous S1P. Furthermore, our results suggest that

endogenous and exogenous S1P are involved in the development

of myogenic tone.

In control experiments, there were no significant differences

between two consecutive pressure-induced myogenic responses

(data not shown). In addition, to determine the EC50 for S1P, SKI,

and NaF, we generated a concentration-response curve for these

drugs at a 80 mmHg transmural pressure. The EC50 values were

1.28 mM for S1P, 5.49 mM for SKI, and 1.01 mM for NaF (data

not shown).

To determine the role of endothelium on endogenous and

exogenous S1P-induced myogenic tone, we compared myogenic

tone between the presence and absence of endothelium in isolated

rabbit PCAs. As shown in Figure 2D, raising the transmural

pressure from 40 to 80 mmHg developed myogenic tone and

pressure-induced myogenic tone was similar between endothelium

denuded (27.1 6 5.8, n= 5) and intact (29.7 6 5.0, n= 5) arteries.

Exogenous S1P-induced myogenic tone was also similar between

endothelium denuded (63.9 6 5.9, n= 5) and intact (66.7 6 7.5,

n = 5) arteries. SKI (1 mmol/L) significantly (P,0.05, n = 5)

inhibited the pressure-induced development of myogenic tone

and the inhibitory effect was not different between endothelium

denuded and intact arteries. However, SKI had no significant

effect on S1P-induced myogenic tone in endothelium denuded and

intact arteries (Figure 2D; n= 5).

2.3. Mechanisms Underlying Endogenous and ExogenousS1P-induced Myogenic ToneWe first determined the influence of S1P on the changes in

Ca2+ concentration by measuring vascular wall Ca2+ concen-

tration using Fura-2AM, the fluorescence ion indicator. As

shown in Figure 3, raising the transmural pressure from 40 to

80 mmHg increased Ca2+ fluorescence intensity. Application of

exogenous S1P further increased Ca2+ fluorescence intensity.

The increase in Ca2+ fluorescence intensity was accompanied by

the development of myogenic tone (Figure 3A1; n = 6). To

determine whether blockading SK1, the S1P-generating en-



zyme, with SKI altered the Ca2+ fluorescence intensity, we

measured the effect of SKI pretreatment on changes in Ca2+

fluorescence intensity. SKI (1 mmol/L) significantly (P,0.05,

n = 6) inhibited the pressure-induced increase in Ca2+ fluores-

cence intensity and development of myogenic tone (Figures 3A2

and B1). However, SKI had no significant effect on the S1P-

induced increase in Ca2+ fluorescence intensity and myogenic

tone (Figures 3A2 and B2; n = 5).

To assess the possible involvement of Ca2+ sensitization of the

contractile apparatus (Ca2+ sensitization) pathway, we evaluated

the role of RhoA/Rho kinase and NADPH oxidase/reactive

oxygen substrate (ROS) on endogenous and exogenous S1P-

induced myogenic tone (Figure 4). Fasudil (1 mmol/L), the Rho

kinase inhibitor, significantly (P,0.05, n= 5) inhibited pressure-

and exogenous S1P-induced myogenic tone (Figures 4B1 and

B2). Inhibition of NADPH oxidase with 10 mmol/L DPI,

a broad chemical inhibitor of signaling pathways unrelated to

ROS generation, significantly (P,0.05, n = 6) reduced pressure-

and exogenous S1P-induced myogenic tone (Figures 4A, C1 and

C2). Furthermore, apocynin, a specific chemical inhibitor of

NADPH oxidase, also significantly (P,0.05, n= 5) reduced

pressure- and exogenous S1P-induced myogenic tone

(Figures 4D1 and D2).

2.4. S1P Receptors Mediate the Effects of Exogenous andEndogenous S1PThree S1P receptors (S1P1–3; formerly known as Edg1, Edg5,

and Edg3) are expressed in rabbit PCA (Figure 5A). These S1P

receptors are also expressed in BAs, MCAs, and ICAs. To

determine which S1P receptors are involved in endogenous and

exogenous S1P-induced changes in myogenic tone, we evaluated

myogenic tone in isolated rabbit PCAs in the presence and

absence of specific S1P receptor blockers. As shown in

Figures 5B and C, pretreatment with 1 mmol/L W146 (S1P1blocker; Figure 5C1; n = 6), 1 mmol/L JTE013 (S1P2 blocker;

Figure 5C2; n = 6), and 1 mmol/L CAY10444 (S1P3 blocker;

Figure 5C3; n= 5) significantly (P,0.05) inhibited not only

pressure-induced myogenic tone, but also S1P-induced myogen-

ic tone. The extent of inhibition was similar among the S1P

receptor blockers.

2.5. 20 kDa Myosin Light Chain ( MLC20) Phosphorylationand Myogenic ToneTo determine the possible downstream effectors of myogenic

tone, we measured MLC20 phosphorylation in steady-state

myogenic tone. As shown in Figure 6, MLC20 phosphorylation

increased significantly (P,0.05, n= 5) when the transmural

pressure was raised from 40 to 80 mmHg. The level of MLC20

phosphorylation induced by pressure was similar to the high K+-

induced MLC20 phosphorylation level. However, the pressure-

induced increase in MLC20 phosphorylation was significantly

inhibited (P,0.05, n = 5) by pre-treatment of the arteries with

SKI. The pressure-induced increase in MLC20 phosphorylation

was further increased by application of exogenous S1P when the

pressure-induced myogenic tone had stabilized. SKI also signifi-

cantly (P,0.05, n = 5) reduced the exogenous S1P-induced

increase in MLC20 phosphorylation. However, the pressure-

induced increase in MLC20 phosphorylation in arteries treated

with S1P and SKI was higher than that in arteries treated with

SKI alone.

Figure 1. Expression and activation of SK1 by elevated of transmural pressure. A: Western blot analysis of SK1 expression in rabbit vessels.Representative western blots of SK1 and actin in isolated rabbit posterior cerebral arteries (PCAs), basilar arteries (BAs), middle cerebral arteries(MCAs), and internal carotid arteries (ICA). B: SK1 iImmunofluorescence in isolated rabbit posterior cerebral arteries at 40 and 80 mmHg. C: SK1immunofluorescence at pressure elevation (80 mmHg) in the absence of external Ca2+ (C1) and in the presence of mechanosensitive ion channel (C2),and voltage-sensitive Ca2+ channel blockers (C3).doi:10.1371/journal.pone.0035177.g001



Discussion

A previous report indicated that the SK1, S1P-generating

enzyme, can function as a mechanotransducer [12–14]. However,

whether the SK1/S1P pathway was involved in myogenic

vasoconstriction under normal physiological conditions was not

determined. Furthermore, mechanisms underlying SK1/S1P

pathway-induced myogenic tone were not fully elucidated.

The results of the present study suggest that the SK1/S1P

pathway plays an important role in myogenic tone. The major

findings of this study are as follows: 1) SK1 is expressed and

activated in response to transmural pressure elevation in rabbit

PCAs. Translocation of SK1 is dependent on Ca2+ influx via

activation of voltage-sensitive Ca2+ channels following depolariza-

tion by activation of mechanosensitice ion channels; 2) pressure-

induced myogenic tone is inhibited in rabbit PCAs treated with

sphingosine kinase inhibitor (SKI), but augmented by treatment

with NaF, an inhibitor of SPP1, NaF; 3) exogenous S1P further

augments the pressure-induced myogenic response; 4) pressure

induces an increase in Ca2+ concentration with the development of

myogenic tone, and the increased Ca2+ concentration and

myogenic tone is inhibited by SKI. Exogenous S1P further

increases the pressure-induced increase in Ca2+ concentration and

myogenic tone, but SKI has no effect. 5) pressure- and exogenous

S1P-induced myogenic tone are inhibited by pre-treatment with

fasudil, Rho kinase inhibitor, and NADPH oxidase inhibitors such

as DPI and apocynin; 6) pressure- and exogenous S1P-induced

myogenic tone are inhibited by pre-treatment with S1P receptor

blockers W146 (S1P1 receptor blocker), JTE013 (S1P2 receptor

blocker), and CAY10444 (S1P3 receptor blocker); and 7) MLC20

phosphorylation increases when transmural pressure is raised from

40 to 80 mmHg, while exogenous S1P further increases

Figure 2. The effect of endogenous and exogenous S1P on myogenic tone. A: Representative traces showing the effect of 5 mmol/L SKI onpressure-induced myogenic tone. B: The mean data for the effects of SKI (B1; n = 9), NaF (B2; n = 6), and S1P (B3; n = 5) on pressure-induced myogenictone. Changes in the lumen diameter were measured in response to 20 mmHg stepwise increases in transmural pressure in Ca2+-containing Kreb’s-Henseleit solution (active tone; KH) or Ca2+-free KH solution (passive tone). C: Representative traces from five independent results showing the effectof pre-treatment of S1P on SKI-induced inhibitory effect of myogenic tone. D: Summarized data for changes in myogenic tone during the elevation oftransmural pressure (from 40 mmHg to 80 mmHg) and application of S1P in posterior cerebral arteries with and without endothelium. S1P andinhibitors were administered at a transmural pressure of 40 mmHg 30 min before increasing in luminal pressure. Data are expressed as means 6SEMs (n = 5–9) and are normalized to myogenic tone at a pressure of 40 mmHg. *Significantly different compared to the control (P,0.05). TMP:transmural pressure. EC: endothelial cells.doi:10.1371/journal.pone.0035177.g002



phosphorylation. The pressure-induced increase in MLC20

phosphorylation is inhibited by pre-treatment of arteries with

SKI. Taken together, these results suggest that the SK1/S1P

pathway may play an important role in pressure-induced

myogenic responses in rabbit PCAs under normal physiological

conditions.

To determine whether endogenous S1P generated in response

to pressure was involved in myogenic vasoconstriction under

normal physiological conditions, we first determined whether SK1

was expressed and activated by pressure in isolated rabbit PCA

using western blotting and immunostaining. We detected SK1

proteins in isolated vessels including PCAs, BAs, MCAs, and ICAs

and found that SK1 was translocated from the cytosol to the

plasma membrane as a result of an increase in transmural pressure

from 40 to 80 mmHg. SK1 translocation from the cytosol to the

plasma membrane is an accepted feature of SK1 activation

[12,22]. To determine the mechanism involved in the trans-

location of SK1 by pressure elevation, we determined whether

SK1 was translocated under several conditions. Translocation of

SK1 by pressure elevation was blocked in the absence of external

Ca2+ and in the presence of mechanosensitive ion channel

blockers, amiloride (epithelial Na+ channel blocker) [23], 9-

phenanthrol (selective TRPM4 blocker) [24], and SKF 96365

(nonselective blocker of TRPC channels) [25]. Translocation of

SK1 by pressure elevation was also blocked in the presence of

nifedipine, voltage-sensitive Ca2+ channel blocker. Although the

blockers used in this study have many non-specific effects, the

concentration used in the present study was reported to have no

non-specific effect. Therefore, our results suggest that SK1 activity

increases after membrane depolarization and Ca2+ influx via

activation of voltage-sensitive Ca2+ channels, and are consistent

with the previous findings that depolarization induces rapid and

transient formation of intracellular S1P [26].

To determine whether endogenous S1P generated by pressure

elevation plays a role in myogenic response, we evaluated the

effect of SKI, an inhibitor of SK1, and NaF, an inhibitor of SPP1,

on the pressure-induced myogenic response in rabbit PCA.

Pressure-induced myogenic tone was inhibited in rabbit PCAs

treated with SKI, but augmented by treatment with NaF. We also

investigated the effect of exogenous S1P on myogenic tone to

Figure 3. Changes in the Ca2+ fluorescence ratio and lumen diameter during transmural pressure elevation and S1P application. A:Representative traces demonstrate changes in the Ca2+ ratio and lumen diameter during the elevation of transmural pressure (from 40 to 80 mmHg)and application of S1P in the absence (A1) or presence (A2) of SKI. B: Summarized data for changes in the Ca2+ ratio and myogenic tone during theelevation of transmural pressure (B1) and application of S1P (B2) in the absence or presence of SKI. Data are expressed as means 6 SEMs (n = 6). TMP:transmural pressure.doi:10.1371/journal.pone.0035177.g003



confirm the role of S1P in the myogenic response. Exogenous S1P

further augmented the pressure-induced myogenic response. We

also determined the effect of pre-treatment of exogenous S1P on

the SKI-induced inhibitory effect of myogenic tone. Pre-treatment

of S1P decreased blood vessel diameter and abolished SKI-

induced inhibitory effect of myogenic tone. Taken together, our

results suggest that the elevation of transmural pressure activates

SK1 and then increases endogenous S1P. Furthermore, our results

suggest that endogenous S1P generated by pressure elevation is

involved in the development of myogenic tone in PCAs and are

consistent with the previous findings that SK1/S1P plays a specific

role as a modulator of cerebral blood flow [27].

It is well known that endothelial cells modulate vascular tone by

releasing nitric oxide. Interestingly, it has previously reported that

SK1 increases nitric oxide production by activation of endothelial

nitric oxide synthase [28]. In the present study, to determine the

role of endothelium on the SK1/S1P-induced myogenic tone, we

measured endogenous and exogenous S1P-induced myogenic tone

in PCAs with and without endothelial cells. We found that

pressure- and S1P-induced myogenic tone were not different

Figure 4. Effects of Rho kinase and NADPH oxidase inhibitors on pressure- and exogenous S1P-induced myogenic tone. A:Representative traces demonstrate changes in lumen diameter during the elevation of transmural pressure (from 40 to 80 mmHg) and application ofS1P in the absence or presence of DPI. B: Summarized data for changes in myogenic tone during the elevation of transmural pressure and applicationof S1P in the absence or presence of fasudil (B; n = 5), DPI (C; n = 6), or apocynin (D; n = 5). Data are expressed as means 6 SEMs (n = 5–6). TMP:transmural pressure.doi:10.1371/journal.pone.0035177.g004



between endothelium denuded and intact arteries. We also found

that the SKI-induced inhibitory effect of myogenic tone was

similar in endothelium denuded and intact arteries.

It is generally accepted that myogenic vasoconstriction is

mediated by a combination of elevation of cytosolic Ca2+

concentration ([Ca2+]i) [29,30] and Ca2+ sensitization [20]. We

have previously reported that S1P-induced vasoconstrictions are

mediated by a combination of Ca2+ mobilization from the

sarcoplasmic reticulum and Ca2+ influx through L-type Ca2+

channels in addition to a Ca2+ sensitization mechanism [19]. In

the present study, to evaluate whether increases in [Ca2+]i and/or

the Ca2+ sensitization pathway contribute to the endogenous S1P-

induced myogenic tone, we first measured the effect of SKI on

changes in [Ca2+]i and myogenic tone when the transmural

pressure was elevated. We found that pressure induced an increase

in [Ca2+]i with subsequent development of myogenic tone, and the

increased [Ca2+]i and myogenic tone were inhibited by SKI. We

also showed that exogenous S1P further increased the pressure-

induced increased [Ca2+]i and myogenic tone, but SKI had no

effect. These results suggest that the increase in [Ca2+]i involved in

myogenic vasoconstriction is mediated by endogenous S1P

generated in response to pressure elevation. However, we did

not determine the source of the [Ca2+]i increase induced by

endogenous S1P.

We investigated the role of the Ca2+ sensitization mechanism

in endogenous S1P-induced myogenic tone, using fasudil, DPI,

and apocynin to assess the influence of Rho A/Rho kinase and

NADPH oxidase-dependent generation of ROS. Fasudil inhib-

ited pressure- and exogenous S1P-induced myogenic tone. DPI

and apocynin also inhibited pressure- and exogenous S1P-

induced myogenic tone. These results suggest that the RhoA/

Rho-kinase and ROS-mediated Ca2+ sensitization mechanisms

play an important role in S1P-induced myogenic tone. Our

results are consistent with the previous findings that S1P-

mediated activation of the RhoA/Rho kinase pathway is an

integral part of myogenic tone [13] and NADPH oxidase-derived

ROS production is increased in response to the elevation of

transmural pressure [31].

Figure 5. Expression of S1P receptors and effects of S1P receptor blockers on pressure- and exogenous S1P-induced myogenictone. A: Western blot analysis of S1P receptor expression in rabbit vessels. Representative western blots of S1P receptors and actin in isolated rabbitposterior cerebral arteries (PCAs), basilar arteries (BAs), middle cerebral arteries (MCAs), and internal carotid arteries (ICA). B: Representative tracesdemonstrate changes in lumen diameter during the elevation of transmural pressure (from 40 to 80 mmHg) and application of S1P in the absence orpresence of JTE013. C: Summarized data for changes in myogenic tone during the elevation of transmural pressure and application of S1P in theabsence or presence of W146 (C1; n = 6), JTE013 (C2; n = 6), and CAY10444 (C3; n = 5). Data are expressed as means 6 SEMs (n = 5–6). TMP: transmuralpressure.doi:10.1371/journal.pone.0035177.g005



Although a link between SK1/S1P and Rho A/Rho kinase or

NADPH oxidase has been identified, we did not identify the specific

signaling mechanisms that allow for their connection. S1P is

a pleiotropic mediator and can act as both an intracellular second

messenger and an extracellular ligand. The exact signaling targets of

intracellular S1P remain unidentified but extracellular S1P signals

are transducedby fivedistinctG-protein coupled receptors (S1P1–5),

which can activate small GTPases (e.g., RhoA and Rac), leading to

NADPHoxidaseactivation[12]. Inthepresent study,weinvestigated

the role of S1P receptors in endogenous and exogenous S1P-induced

myogenic tone. Although five S1P receptors (S1P1–5) have been

identified [32], three specific receptors (S1P1–3) are reportedly

expressed at the mRNA level in vascular smooth muscle [33]. We

verified that S1P1–3 are expressed at the protein level using western

blot. To determine the role of S1P receptors in endogenous and

exogenous S1P-induced myogenic tone, we tested the effects of S1P

receptor specific blockers such as W146 (S1P1 receptor blocker),

JTE013 (S1P2 receptor blocker), and CAY10444 (S1P3 receptor

blocker) on endogenous andexogenousS1P-inducedmyogenic tone.

All three type-specific receptor blockers significantly inhibited both

pressure- (endogenous S1P) and exogenous S1P-induced myogenic

tone. These results suggest that endogenous S1P generated in

response to the elevation of pressure may act not only as an

intracellular second messenger, but also as an extracellular ligand

after being transported across the plasmamembrane. Intracellularly

generated S1P is unable to move through hydrophobic mammalian

cell plasma membranes since it possesses a polar head group.

Although the mechanism of S1P release from cells is not completely

understood, the involvement of the ATP-binding cassette (ABC)

familyof transporters,especiallyABCC7(CFTR),hasbeensuggested

[14].

Smooth muscle contraction is activated primarily by phosphor-

ylation at Ser19 of the 20 kDa regulatory light chain of myosin II.

Therefore, to directly prove that SK1/S1P plays an important role

in pressure-induced myogenic constriction under normal physio-

logical conditions, it is very important to determine the changes in

MLC20 phosphorylation during the development of myogenic

responses and the effect of SK1/S1P blockade on MLC20

phosphorylation. In the present study, we observed an increase

in MLC20 phosphorylation in rabbit PCAs when transmural

pressure or exogenous S1P was applied. The pressure-induced

increase in MLC20 phosphorylation was inhibited by pretreatment

with SKI. Thus, the data suggest that enhanced MLC20

phosphorylation in response to endogenous S1P generated by

transmural pressure elevation plays a central role in the

modulation of myogenic tone.

Based on the results of the present study, it is clear that

endogenous S1P-induced myogenic tone can be regulated by

Ca2+-dependent and/or Ca2+-independent (Ca2+-sensitization)

mechanisms. Increased transmural pressure activates mechanosen-

sitive ion channels that putatively lead to Ca2+ influx via voltage-

sensitive Ca2+ channels and activation of SK1. SK1 converts

sphingosine to S1P. Endogenous and/or exogenous S1P then

increase the intracellular Ca2+ concentration andmyosin light chain

phosphorylation via activation of myosin light chain kinase. On the

other hand, extracellular S1P acts as a receptor ligand and activates

several signaling pathways, including RhoA/Rho kinase and Rac.

RhoA/Rho kinase increases apparent Ca2+ sensitivity by inhibiting

myosin light chain phosphatase (MLCP). The activation of Rac is

associated with increased formation of O2 – via NADPH oxidase.

This pathway also modulates the apparent Ca2+ sensitivity by

inhibiting MLCP.

In summary, our results suggest that the SK1/S1P pathway may

play an important role in pressure-induced myogenic responses in

rabbit PCAs under normal physiological conditions. S1P generated

through SK1 activation by pressure increases myogenic tone. The

underlyingmechanisms forendogenousS1P-inducedmyogenic tone

are an increase in [Ca2+]i and the Ca2+ sensitization mechanism via

Rho A/Rho kinase and NADPH oxidase/ROS. Because endoge-

nous S1P andROSproduction is elevated under pathophysiological

conditions such as hypertension, atherosclerosis, and vasospasm, the

SK1/S1P pathway likely plays an important role in myogenic tone

under pathophysiological conditions.

Author Contributions

Conceived and designed the experiments: YHL ML DSA. Performed the

experiments: ML SKC YEC SIY ECK. Analyzed the data: YHL ML SKC

YEC SIY ECK. Contributed reagents/materials/analysis tools: ML SKC

YEC SIY ECK YHL. Wrote the paper: YHL ML DSA.

Figure 6. Changes in 20-KDa myosin light chain (MLC20) phosphorylation with the elevation of transmural pressure, and the effectsof S1P or SKI application. Results are representative of immunoblots from five independent preparations. Results are expressed as means6 SEMs(n = 5). TMP: transmural pressure.doi:10.1371/journal.pone.0035177.g006



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The Role of Sphingosine Kinase 1/Sphingosine-1- Phosphate ... · Funding: This study was supported by a faculty research grant from Yonsei University College of Medicine in 2010 (No